Development of an HTS assay for Na+, K+-ATPase using nonradioactive rubidium ion uptake
| Autor: | Rajwant Gill, Dong Liang, Sikander Gill, David Wicks, Sasko Despotovski |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
ATPase
Analytical chemistry Drug Evaluation Preclinical chemistry.chemical_element CHO Cells Sensitivity and Specificity Ouabain Rubidium Microtiter plate Spectrophotometry Cricetinae Drug Discovery medicine Animals Humans Na+/K+-ATPase Enzyme Inhibitors Incubation Chromatography biology medicine.diagnostic_test Dose-Response Relationship Drug Spectrophotometry Atomic Dose–response relationship chemistry biology.protein Molecular Medicine Sodium-Potassium-Exchanging ATPase medicine.drug |
| Zdroj: | Assay and drug development technologies. 2(5) |
| ISSN: | 1540-658X |
| Popis: | A high-throughput screening (HTS) assay was developed for the Na(+),K(+)-ATPase channel in order to study rubidium uptake as a measure of the functional activity and modulation of this exchanger. The assay uses elemental rubidium as a tracer for K(+) ions. Three cell lines were used to study the exchanger, and the assay was performed in a 96-well microtiter plate format. Rb(+) uptake was carried by the CHO-K1 cells at 37 degrees C; the maximum ion influx was at 80 min of incubation of the cell line in the medium containing 5.4 mM RbCl. The cells were incubated in Rb(+) uptake buffer (5.4 mM) and with the pump blocker ouabain for 1, 2, and 3 h, respectively. A complete block of the Rb(+) uptake was observed with a 5 mM concentration of ouabain for all the three time intervals. The ouabain 50% inhibitory concentration (IC(50)) value for CHO-K1 cell line ATPase was observed to be 298 microM after 3 h of incubation. In addition, IC(50) values of 94 and 89 microM were observed at 30 min of incubation, indicating that the protocol shows reproducible results. A Z' factor higher than 0.7 was observed in the assays. These studies extend the profile of Na(+),K(+)-ATPases and demonstrate the feasibility of this HTS assay system to screen for compounds that pharmacologically modulate the function of Na(+),K(+)-ATPase. |
| Databáze: | OpenAIRE |
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